The goal of the proposed research is to identify, isolate, and characterize voltage-dependent Na ion channels from mammalian brain. Pinched-off nerve endings (synaptosomes) from rat brain will be used in these experiments because they retain, in vitro, many of the physiological properties of intact nerve endings, in vivo. Na ion channels in synaptosomes will be detected by two methods: depolarization caused by opening Na ion channels will be monitored using a fluorescent probe; influx of radioactive 22Na will be measured by rapid filtration. The regulation of synaptosomal Na ion channels by three classes of neurotoxins, represented by saxitoxin, batrachotoxin, and polypeptide toxins, respectively, will be studied. Various animal venoms will be examined for the presence of new polypeptide toxins that regulate Na ion channels. Using binding of radioactively labelled neurotoxins as an assay, neurotoxin binding sites will be removed from the synaptosomal membranes with detergents and subjected to fractionation by standard protein purification procedures. It is hoped that the use of several neurotoxins as ligands will increase the likelihood of retaining all of the regulatory and transport components of the Na ion channels during purification. At various stages of purification, neurotoxin binding proteins will be incorporated into artificial phospholipid bilayer vesicles (liposomes) in order to reconstitute neurotoxin-regulated Na ion transport activity. Since the Na ion channels and other excitable membrane components are controlled by the membrane potential, the method of monitoring membrane potentials in suspensions of particles using fluorescent probes will be refined for synaptosomes and liposomes in order to improve the method for the study of the regulation of Na ion channels by the membrane potential in these systems.